Thomas C. Caffrey

Nuclear association of the cytoplasmic tail of MUC1 and β-catenin

MUC1, an integral membrane mucin associated with the metastatic phenotype, is overexpressed by most human carcinoma cells. The MUC1 cytoplasmic tail (CT) is postulated to function in morphogenetic signal transduction via interactions with Grb2/Sos, c-Src, and β-catenin. We investigated intracellular trafficking of the MUC1 CT, using epitope-tagged constructs that were overexpressed in human pancreatic cancer cell lines S2-013 and Panc-1. The MUC1 CT was detected at the inner cell surface, in the cytosol, and in the nucleus of cells overexpressing MUC1. Fragments of the MUC1 CT were associated with β-catenin in both cytoplasm and nuclei. Overexpression of MUC1 increased steady state levels of nuclear β-catenin but decreased nuclear levels of plakoglobin (γ-catenin). There was no detectable association between plakoglobin and the MUC1 CT. Coimmunoprecipitation experiments revealed that the cytoplasmic and nuclear association of MUC1 CT and β-catenin was not affected by disruption of Ca2+-dependent intercellular cadherin interactions. These results demonstrate nuclear localization of fragments of MUC1 CT in association with β-catenin and raise the possibility that overexpression of the MUC1 CT stabilizes β-catenin and enhances levels of nuclear β-catenin during disruption of cadherin-mediated cell-cell adhesion.

MUC1 mucin stabilizes and activates hypoxia-inducible factor 1 alpha to regulate metabolism in pancreatic cancer

Aberrant glucose metabolism is one of the hallmarks of cancer that facilitates cancer cell survival and proliferation. Here, we demonstrate that MUC1, a large, type I transmembrane protein that is overexpressed in several carcinomas including pancreatic adenocarcinoma, modulates cancer cell metabolism to facilitate growth properties of cancer cells. MUC1 occupies the promoter elements of multiple genes directly involved in glucose metabolism and regulates their expression. Furthermore, MUC1 expression enhances glycolytic activity in pancreatic cancer cells. We also demonstrate that MUC1 expression enhances in vivo glucose uptake and expression of genes involved in glucose uptake and metabolism in orthotopic implantation models of pancreatic cancer. The MUC1 cytoplasmic tail is known to activate multiple signaling pathways through its interactions with several transcription factors/coregulators at the promoter elements of various genes. Our results indicate that MUC1 acts as a modulator of the hypoxic response in pancreatic cancer cells by regulating the expression/stability and activity of hypoxia-inducible factor-1α (HIF-1α). MUC1 physically interacts with HIF-1α and p300 and stabilizes the former at the protein level. By using a ChIP assay, we demonstrate that MUC1 facilitates recruitment of HIF-1α and p300 on glycolytic gene promoters in a hypoxia-dependent manner. Also, by metabolomic studies, we demonstrate that MUC1 regulates multiple metabolite intermediates in the glucose and amino acid metabolic pathways. Thus, our studies indicate that MUC1 acts as a master regulator of the metabolic program and facilitates metabolic alterations in the hypoxic environments that help tumor cells survive and proliferate under such conditions.

Lipopolysaccharide induces double-stranded DNA fragmentation in mouse thymus : protective effect of zinc pretreatment

Intraperitoneal injection of female NAW/W1 mice with 5 mg of Salmonella typhimurium lipopolysaccharide/kg results in decreased body and thymus weight. Reduced thymic weight is accompanied by fragmentation of DNA into multimers of about 200 bp size. This effect is consistent with the induction of intranucleosomal cleavage of double-stranded DNA in thymus. Maximal fragmentation of DNA occurs between 18 and 24 h after treatment; by 48 h post lipopolysaccharide treatment, there is little evidence of thymic DNA fragmentation. Pretreatment of mice with Zn protects against lipopolysaccharide-induced DNA fragmentation. This effect is maximal at about 72 h after Zn treatment (24 h after lipopolysaccharide treatment) and persists until about 96 h after Zn treatment. At 72 h after pretreatment, the antagonism of thymic DNA fragmentation by Zn is dose-dependent. To examine the role of the acute phase inflammatory response elicited by lipopolysaccharide treatment in the production of changes in thymic weight and DNA integrity, the effects of treatment with casein, a well-characterized inducer of the acute phase inflammatory response in mice, were examined. In contrast to the effect of lipopolysaccharide, casein treatment did not produce a similar pattern of DNA fragmentation in thymus. Taken together, these data suggest that lipopolysaccharide induces DNA fragmentation in thymus by a mechanism which does not occur during the pathophysiological changes which accompany the casein-induced acute phase response. Further, the antagonism by Zn of lipopolysaccharide-induced fragmentation of thymic DNA is consistent with earlier findings that Zn can prevent dexamethasone-induced DNA fragmentation in vitro.

Cyclin-Dependent Kinase 5 Is Amplified and Overexpressed in Pancreatic Cancer and Activated by Mutant K-Ras

Purpose: To evaluate the nature of cyclin-dependent kinase 5 (CDK5) hyperactivity in pancreatic cancer progression. Experimental Design: We used genetic, biochemical, and molecular biology methods to investigate the nature and function of overexpression of CDK5 and its activators p35 and p39 during the progression of pancreatic cancer. Results: Amplification of the CDK5 gene or either of its main activators, p35 and p39, was observed in 67% of human pancreatic ductal adenocarcinoma (PDAC). CDK5, p35, and p39 were rarely expressed in pancreatic ducts whereas more than 90% of PDACs had increased levels of CDK5 and p35. Increased levels of CDK5, p35, and p39 protein were observed in several pancreatic cancer cell lines. Inhibition of CDK5 kinase activity using a CDK5 dominant-negative mutant or the drug roscovitine significantly decreased the migration and invasion of pancreatic cancer cells in vitro. Increased CDK5 kinase activity was also observed in immortalized human pancreatic nestin-expressing (HPNE) cells expressing a mutant form of K-Ras (G12D) compared with HPNE cells expressing native K-Ras. G12D K-Ras increased cleavage of p35 to p25, a stable and greater activator of CDK5, thus implicating a role for CDK5 in early progression of PDAC. Inhibition of the signaling cascade downstream of mutant K-Ras (G12D) that involves mitogen-activated protein/extracellular signal–regulated kinase, phosphoinositide 3-kinase, or CDK5 decreased p25 protein levels. Conclusion: These results suggest that mutant K-Ras acts in concert with CDK5 and its activators to increase malignant progression, migration, and invasion of pancreatic cancer cells. Clin Cancer Res; 17(19); 6140–50. ©2011 AACR.

Tumor-specific immunity in MUC1.Tg mice induced by immunization with peptide vaccines from the cytoplasmic tail of CD227 (MUC1)

Purpose: CD227 (MUC1), a membrane-associated glycoprotein expressed by many types of ductal epithelia, including pancreas, breast, lung, and gastrointestinal tract, is overexpressed and aberrantly glycosylated by malignant cells. We sought to define epitopes on MUC1 recognized by the different cell-mediated immune responses by an in vivo assay. Epitopes identified by this assay were evaluated for efficacy to protect mice transgenic for human MUC1 (MUC1.Tg) against MUC1-expressing tumor growth. Methods: We investigated contributions of the tandem repeat (TR) and the cytoplasmic tail (CT) of MUC1 to the MUC1-specific immunological rejection of tumor cells. MUC1 cDNA constructs, in which the TR region was deleted or the CT was truncated, were transfected into two different murine tumor cell lines (B16 and Panc02), which were used to challenge mice and evaluate immunological rejection of the tumors. We used tumor rejection in vivo to define epitopes on the TR and CT of MUC1 recognized by T cell–mediated immune responses in a preclinical murine model. Results: Our findings demonstrated that the TR and a portion of the MUC1 CT contributed to CD4+ T cell rejection of MUC1-expressing B16 tumor cells, but not rejection of MUC1-expressing Panc02 tumor cells. A separate epitope in the CT of MUC1 was necessary for CD8+ T cell rejection of Panc02 tumor cells. Based on these studies, we sought to evaluate the efficacy of immunizing mice transgenic for (and immunologically tolerant to) human MUC1 with peptides derived from the amino acid sequence of the CT of MUC1. Results showed that survival can be significantly prolonged in vaccinated MUC1.Tg mice challenged with MUC1-expressing tumor cells, without induction of autoimmune responses. Conclusions: These studies demonstrated that MUC1 peptides may be utilized as an effective anticancer immunotherapeutic, and confirmed the importance of immunogenic epitopes outside of the TR.

MUC1 regulates cyclin D1 gene expression through p120 catenin and β-catenin.

MUC1 interacts with β-catenin and p120 catenin to modulate WNT signaling. We investigated the effect of overexpressing MUC1 on the regulation of cyclin D1, a downstream target for the WNT/β-catenin signaling pathway, in two human pancreatic cancer cell lines, Panc-1 and S2-013. We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1. In stark contrast, cyclin D1 promoter activity was not affected in moderately differentiated S2-013.MUC1F cells that overexpressed recombinant MUC1 relative to S2-013.NEO cells that expressed low levels of endogenous MUC1. The S2-013 cell line was recently shown to be deficient in p120 catenin. MUC1 is known to interact with P120 catenin. We show here that re-expression of different isoforms of p120 catenin restored cyclin D1 promoter activity. Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso. Thus, full activation of cyclin D1 promoter activity requires β-catenin activation of TCF-lef and stabilization of specific p120 catenin isoforms to relieve the repression of KAISO. Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

Impact of MUC1 mucin downregulation in the phenotypic characteristics of MKN45 gastric carcinoma cell line.

Background Gastric carcinoma is the second leading cause of cancer-associated death worldwide. The high mortality associated with this disease is in part due to limited knowledge about gastric carcinogenesis and a lack of available therapeutic and prevention strategies. MUC1 is a high molecular weight transmembrane mucin protein expressed at the apical surface of most glandular epithelial cells and a major component of the mucus layer above gastric mucosa. Overexpression of MUC1 is found in approximately 95% of human adenocarcinomas, where it is associated with oncogenic activity. The role of MUC1 in gastric cancer progression remains to be clarified. Methodology We downregulated MUC1 expression in a gastric carcinoma cell line by RNA interference and studied the effects on cellular proliferation (MTT assay), apoptosis (TUNEL assay), migration (migration assay), invasion (invasion assay) and aggregation (aggregation assay). Global gene expression was evaluated by microarray analysis to identify alterations that are regulated by MUC1 expression. In vivo assays were also performed in mice, in order to study the tumorigenicity of cells with and without MUC1 downregulation in MKN45 gastric carcinoma cell line. Results Downregulation of MUC1 expression increased proliferation and apoptosis as compared to controls, whereas cell-cell aggregation was decreased. No significant differences were found in terms of migration and invasion between the downregulated clones and the controls. Expression of TCN1, KLK6, ADAM29, LGAL4, TSPAN8 and SHPS-1 was found to be significantly different between MUC1 downregulated clones and the control cells. In vivo assays have shown that mice injected with MUC1 downregulated cells develop smaller tumours when compared to mice injected with the control cells. Conclusions These results indicate that MUC1 downregulation alters the phenotype and tumorigenicity of MKN45 gastric carcinoma cells and also the expression of several molecules that can be involved in tumorigenic events. Therefore, MUC1 should be further studied to better clarify its potential as a novel therapeutic target for gastric cancer.

Duct epithelial cells cultured from human pancreas processed for transplantation retain differentiated ductal characteristics

A procedure is described for the isolation and growth in vitro of epithelial cells from the duct network of human pancreas, referred to as DEC. A significant advantage of our procedure over previously published procedures is that it enables the isolation of DEC from small pieces of pancreas tissue (<5 g) and, also, from the digest remaining after the isolation of islet cells from human pancreas, material that would normally be discarded. These were the only reliable sources for pancreas tissue available to us. This procedure shows that some of the techniques that have been successfully used for the isolation of rodent DEC are also valuable in the isolation of human DEC. In particular, the use of cholera toxin to prevent fibroblast growth and contamination obviates the need for the time-consuming procedure of physically removing fibroblasts or the use of expensive fibroblast-specific monoclenal antibodies. The use of sieving to separate the digest immediately achieves a partial purification, which, coupled with that of allowing duct cysts to form, adds to the purity of the final preparation. The ductal system of the intact pancreas tissue and the DEC derived from it expressed cytokeratins 7, 8/18, and 19 and markers for the presence of MUCl, CFTR, and carbonic anhydrase II, which are specific for ductal epithelial cells or for pancreatic ductal functions. This study showed that it is possible to obtain selectively viable DEC from small ducts in otherwise waste pieces of human pancreas. It showed that these cells retained all of the epithelial characteristics that were examined and, in combination with data from an earlier study, showed that the cultured DEC retain the metabolic functions of duct epithelial cells in vivo.

Effect of sodium butyrate on metallothionein induction and cadmium cytotoxicity in ROS 17/2.8 cells

ROS 17/2.8 cells, a cloned rat osteosarcoma cell line, are exceptionally sensitive to the cytotoxic effects of cadmium. This sensitivity is associated with the inability of this metal to induce the synthesis of metallothionein, a transition metal-binding protein, which detoxifies this metal by its sequestration. Sodium butyrate induces the synthesis of metallothionein in these cells in a concentration-dependent manner. Treatment with this agent also significantly increases the resistance of these cells to the cytotoxic effects of cadmium and the protective effect of butyrate is reversed upon its removal from culture medium. Butyrate treatment did not significantly alter the accumulation of cadmium by these cells. Hence, the increased synthesis of metallothionein in butyrate-treated cells is not due to increased cellular uptake of cadmium. Inhibition of DNA synthesis due to butyrate was not a sufficient condition to alter metallothionein synthesis or to protect against Cd-induced cytotoxicity. Equivalent inhibition of DNA synthesis with hydroxyurea failed to increase metallothionein synthesis in cadmium-treated cells. These results indicate that modulation of metallothionein gene expression in this cell line is the critical factor in determining cellular sensitivity to the cytotoxic effects of cadmium.

SOX2 functions as a molecular rheostat to control the growth, tumorigenicity and drug responses of pancreatic ductal adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly deadly malignancy. Expression of the stem cell transcription factor SOX2 increases during progression of PDAC. Knockdown of SOX2 in PDAC cell lines decreases growth in vitro; whereas, stable overexpression of SOX2 in one PDAC cell line reportedly increases growth in vitro. Here, we reexamined the role of SOX2 in PDAC cells, because inducible SOX2 overexpression in other tumor cell types inhibits growth. In this study, four PDAC cell lines were engineered for inducible overexpression of SOX2 or inducible knockdown of SOX2. Remarkably, inducible overexpression of SOX2 in PDAC cells inhibits growth in vitro and reduces tumorigenicity. Additionally, inducible knockdown of SOX2 in PDAC cells reduces growth in vitro and in vivo. Thus, growth and tumorigenicity of PDAC cells is highly dependent on the expression of optimal levels of SOX2 – a hallmark of molecular rheostats. We also determined that SOX2 alters the responses of PDAC cells to drugs used in PDAC clinical trials. Increasing SOX2 reduces growth inhibition mediated by MEK and AKT inhibitors; whereas knockdown of SOX2 further reduces growth when PDAC cells are treated with these inhibitors. Thus, targeting SOX2, or its mode of action, could improve the treatment of PDAC.